1. Field of the Invention
The present invention relates to bis-biotin compounds and compositions that find use, for example, in assays for analytes, such as, e.g., immunoassays, receptor assays and nucleic acid assays. In such assays it is often necessary to bind together two components, one being, for example, a specific binding member and the other being another assay component such as, for example, surfaces, antigens, haptens, nucleic acids, proteins such as antibodies, etc.
It has been found that reagents containing biotin are convenient for use in such assays. Such biotin reagents generally have one of the components to be bound conjugated to biotin. An avidin reagent is also employed that has avidin or streptavidin bound to the other of the components to be bound. To bring about binding of the two components, it is merely necessary to combine the biotin reagent with the avidin reagent. The binding interactions between biotin and the biotin binding site of avidin are the result of, among others, formation of multiple hydrogen bonds and van der Waals interactions between biotin and avidin together with the ordering of surface polypeptide loops that bury the biotin in the protein interior.
However, despite the strong binding of free biotin to avidin, biotin that is conjugated to another molecule does not bind as strongly. Assay components bound through biotin-avidin bonds can dissociate relatively rapidly during standing in dilute solutions or upon washing a surface to which they are attached. This phenomenon becomes particularly problematic in homogeneous assays where conjugates of specific binding pair members with labels may be stored in dilute solution for long periods prior to use.
Accordingly, there is a need for a binding pair that permits labeled assay components to bind to each other on mixing so efficiently that there is no free component that must be separated from the bound and there is no tendency of the bound component to dissociate even on standing for long periods of time.
Pairs of biotin molecules have previously been linked to each other through chains of varying lengths. There are reports of both monofunctional, namely, binding of one biotin, and bifunctional, namely, binding of both biotins, to avidin or streptavidin. Bis-biotins have been used to create ordered arrays of streptavidin through the formation of linear polymers of avidin or streptavidin. In situations where binding of bis-biotins has been bifunctional, indications are that only relatively weak binding was obtained. Multiple biotins have been attached to various sbp members. Usually, the preparations are random both with regard to the number and placement of the biotins, and most preparations utilize the sbp member as part of the chain linking the biotins. Generally, multiple biotins are attached because of the difficulty of attaching exactly one biotin or the opportunity to subsequently affix multiple labels through binding of multiple avidin molecules. Multiple biotins have been added to one terminus of an oligonucleotide because the biotins can be readily added by chain extension using terminal transferase. Also, avidin binds more efficiently at the oligonucleotide termini. In one case two biotins were deliberately introduced into an oligonucleotide at specific sites. The observed numbers of streptavidins that bound appeared to be additive and, thus, did not suggest bivalent binding.
2. Description of the Related Art
U.S. Pat. No. 4,298,685 (Parikh, et al.) discloses a diagnostic reagent for use in assays. The reagent is a conjugate of biotin and antibodies for the substance to be determined in the assay.
Hapten-biotin conjugates are discussed in U.S. Pat. No. 5,219,764 (Huber, et al.). In such conjugates the hapten is linked to biotin by means of a spacer, which has 26 to 40 atoms in its chain and contains at least 5 heteroatoms. The conjugates find use in certain homogeneous immunoassays.
Green discusses avidin and streptavidin complexes in Methods in Enzymology (1990) 184:51-67.
Green, et al., disclose the use of bifunctional biotinyl compounds to determine the arrangement of subunits in avidin (Biochem. J. (1971) 125:781-791).
Structural origins of high-affinity biotin binding to streptavidin is discussed by Weber, et al., in Science (1989) 243:85-88.
Morgan, et al., describe the polymerization of avidin and streptavidin with aromatic bis-biotin ligands in Polym. Sci., Part A: Polym. Chem. (1994) 32:1331.
Pierlot, et al., discloses solid phase synthesis of 5' non-radioactive multiple labeled oligodeoxyribonucleotides in Bioorg. Med. Chem. Lett. (1992) 2:267-271.
Akiyama discloses bis-biotinylated endothelin in Protein Expression Purif. (1992) 3:427.
The synthesis and structural characterization of a cardioactive biotinylated digoxin analogue is disclosed by Nutikka in Clin. Bioch. (1991) 24:469-473.